Recent advances in genetic research, especially those focused upon cancer or inherited disorders, has led to the identification of new genes having specific patterns of DNA sequence alterations directly related to pathologic disease states (Weinberg R A, Oncogenes, Antioncogenes, and the Molecular Basis of Multistep Carcinogenesis. Cancer Res., 49:3713–3721, 1989). Central to this research is the Human Genome Project, a monumental world-wide scientific effort to fully map and sequence the human genome (Watson J D, The Human Genome Project: Past, Present, and Future. Science, 248:44–49, 1990). Together the results will provide a detailed blueprint of the normal Human genome together with a understanding of DNA damage upon which the diagnosis and treatment of many conditions may be formulated. Using this information, genetic based therapies have already been instituted, consisting of the introduction into selected cells of normal or modified human genes designed to integrate and function as part of the host genome (Anderson W F, Human Gene Therapy. Science, 256:808–813, 1992). These initiatives provide a strong stimulus for tissue based methods which can characterize in detail DNA sequence alterations in selected cellular components of normal and disease affected human tissues.
To realize the potential of the expanding database of DNA sequence information, it has become necessary to have available methods which can detect and characterize DNA sequence alterations in tissue specimens such as those routinely obtained during the medical management of patients. Presently, in clinical practice, genetic analysis usually requires a fresh and relatively large tissue sample secured independently of other specimens for diagnostic purposes. Realistically, many clinical specimens, for which genetic sequence information would be vitally needed, are of small size obtained through biopsy procedures. Moreover there exists a priority of tissue management in that proper histopathologic diagnosis is paramount demanding that adequate tissue first be secured and placed into appropriate fixative solutions to preserve morphologic integrity for accurate is histopathologic evaluation. Standard practices of genetic analysis are generally ineffective on specimens exposed to fixative agents (Ben Ezra J, Johnson D A, Rossi J, Cook N, Wu A, Effect of Fixation on the Amplification of Nucleic acids from Paraffin-Embedded Material by the Polymerase Chain Reaction. J. Histochem. Cytochem., 39:351–354, 1991). This has led some to the collection of fresh tissue in Freezer Banks, a process that is inconvenient, costly and very often not carried out in practice. In contrast stands the vast bulk of archival tissue specimens in the form of fixative treated, paraffin embedded tissue blocks. These tissue archives are present in all medical centers and contain valuable specimens from patients whose disease has been followed for many years as part of the normal clinical management. These informative specimens await a simple, effective means for their genetic analysis. Clinical practice is very much in need of easily applicable techniques for DNA sequence analysis from routinely prepared tissue blocks (Antonarakis S E, Diagnosis of Genetic Disorders at the DNA Level. N. Engl. J. Med., 320:153–163, 1989). The methods to be used should not be destructive to the blocks and must take advantage of the important insights gained through detailed histopathologic analysis. The techniques should be independent of archival storage time permitting the opportunity for long-term retrospective study. Finally the approach should be cost effective and timely to participate in everyday clinical decision making.
Topographic Genotyping (TG) is a novel system of tissue management comprehensive in scope specifically developed to address these specific issues enabling full DNA analysis within the context of traditional pathology. TG permits tissue specimens, routinely fixed in standard fixative chemical agents, of any size including minute needle biopsy specimens and cell blocks of cytology material, and of any age including those stored in paraffin for over thirty years, to be both fully available for standard histopathology examination as well as DNA sequence analysis. Furthermore, TG has been specifically designed to incorporate procedures for tissue and information handling allowing quick and easy clinical as well as research application. In essence TG is designed to allow the user to simply and effectively sample minute morphologic targets within fixative treated tissue specimens based on histopathologic and topographic considerations, which in turn may serve as the basis for detailed DNA sequence analysis. The results of TG is an integration of genetic and histopathologic features in a simple, reliable and cost effective manner for clinical application.
Solid tissue specimens, removed at surgery or through biopsy procedures, are exposed to fixative agents designed to prevent tissue breakdown and preserve morphologic integrity for microscopic analysis and archival storage. Fixatives, the most common being a 4% buffered solution of formaldehyde, cause their tissue preserving effect by a process of chemical crosslinking of cellular constituents including proteins, sugars and nucleic acids. Much of the tissue stabilizing effect of tissue fixatives is chemically irreversible (Greer C E, Oeterson S L, Kiviat N B, Manos M M, PCR Amplification from Paraffin-Embedded Tissues. Effect of Fixative and Fixation Time. Am. J. Clin. Path., 95:117–124, 1991). This tissue stabilizing and preserving chemical interaction, essential for microscopic analysis, greatly interferes with the manipulation of DNA for genetic investigation representing a major deterrent for general application of molecular analysis on fixed tissue specimens.
In order to meet the need for up to date genetic analysis, current medical practice recommends obtaining separate tissue specimens, not subject to chemical fixation, exclusively for the purpose of genetic analysis. When this involves a fluid specimen of homogeneous character such as a blood sample or bone marrow aspirate, division of the specimen for separate microscopic and molecular biologic analysis is usually accomplished fairly easily without involving interfering with traditional pathologic diagnosis. For many needle biopsy procedures, however, and in a variety of other circumstances of limiting tissue availability, a solid tissue sample will not be able to be appropriately divided and thus molecular examination would not be performed. Even in the case of large specimens, which might appear at first to provide generous amounts of tissue sample for genetic study, appropriate subdivision may not be feasible in as much as cellular heterogeneity cannot be fully appreciated until full tissue fixation and histopathologic examination is first performed. To derive the greatest benefit from genetic analysis, it is highly desirable to focus molecular analysis on selected tissue targets reflecting the cellular basis of disease processes. This in turn can only be achieved following thorough histopathologic examination. This is the essential condition that must be met if true and effective integration of pathology and molecular biology is to be achieved.
These realities provide a strong impetus to define new ways in which fixative treated tissue specimens should be handled to allow DNA structure analysis. Current protocols in this regard, while they may be available, are, in general, highly inefficient, difficult to apply widely in clinical practice and do not take histological considerations fully into account (Shibata D K, Arnheim N, Martin W J, Detection of Human Papilloma Virus in Paraffin-Embedded Tissue Using the Polymerase Chain Reaction. J. Exp. Med., 167:225–230, 1988; Wright D K, Manos M M, Sample Preparation from Paraffin-Embedded Tissues. in PCR Protocols: A Guide to Methods and Applications. Innes M A, Gelfand D H, Sninsky J J, White T J (eds). pp. 153–158, 1990, Academic Press, Berkeley, Calif.; Greer C E, Lund J K, Manos M M, PCR Amplification from Paraffin-Embedded Tissues: Recommendations on the Fixatives for Long-Term Storage and Prospective Studies. PCR Meth. & Applic. 1:46–50, 1991). There exists at present no systematic means to integrated morphologic and genetic analysis of solid tissue specimens. Authoritative sources have recommended a system of DNA extraction and precipitation analogous to that used with fresh tissue (Shibata D K, Arnheim N, Martin W J, Detection of Human Papilloma Virus in Paraffin-Embedded Tissue Using the Polymerase Chain Reaction. J. Exp. Med., 167:225–230, 1988). Nucleic acid precipitation from fixative treated specimens is very inefficient with resultant low recovery yields for subsequent genetic analysis (Ben Ezra J, Johnson D A, Rossi J, Cook N, Wu A, Effect of Fixation on the Amplification of Nucleic acids from Paraffin-Embedded Material by the Polymerase Chain Reaction. J. Histochem. Cytochem., 39:351–354, 1991). This in turn demands sacrifice of large amounts of starting material which is impractical and highly undesirable in many instances. Genetic analysis of fixative treated tissues often require gross dissection of the paraffin block in the dry state, a procedure that is both uncontrolled, wasteful and destructive to the archival stored tissue. This approach fails to take into account important microscopic features since handling of the tissue in the paraffin block with the naked eye is not subject to fine microscopic control. Protocols direct one to scrape tissue off the slide with a scalpel. occasionally protocols suggest selecting the area according to structures seen using the human eye (not microscope) similarly ignore the benefits derived from careful histopathologic/topographic selection (Greer C E, Lund J K, Manos M M, PCR Amplification from Paraffin-Embedded Tissues: Recommendations on the Fixatives for Long-Term Storage and Prospective Studies. PCR Meth. & Applic. 1:46–50, 1991). Tissue sampling must be performed at the microscopic level responding to the unique topographic and histopathologic features that are present in an individual tissue specimen.
DNA extracted from fixative treated tissues is generally regarded as a relatively poor starting material for nucleic acid amplification, mutational analysis and DNA sequencing (Ben Ezra J, Johnson D A, Rossi J, Cook N, Wu A, Effect of Fixation on the Amplification of Nucleic acids from Paraffin-Embedded Material by the Polymerase Chain Reaction. J. Histochem. Cytochem., 39:351–354, 1991). Users are cautioned to this effect and advised to expect poor results. Yet in selected circumstances, these very same specimens may be shown to yield important genetic information indicating that given the right approach such tissue may be very informative. Despite the knowledge that specific DNA sequence alterations are frequently found involving oncogene and tumor suppressor genes commonly present in many forms of human cancer, there exists at present no effective means to broadly analyze fixative treated specimens of any size and age for clinical application of genetic information.
Topographic genotyping was developed to meet the needs for selection of fixed tissue for genetic analysis. The specific criteria upon which topographic genotyping is based are outlined in Table 1. TG is the only system at present which fully meets these necessary criteria for clinical application. TG furthermore includes the necessary organization of methodological steps and information flow for suitable clinical application at this time. While reports by others document the use of fixative treated tissues for genetic analysis none is uniquely designed to be fully integrated into traditional histopathology in a simple, reliable, efficient and cost effective fashion. Criteria for the topographic component as outline in Table 1 provide the essential link to merge modern genetic analysis into traditional pathology practice.
TABLE 1TOPOGRAPHIC GENOTYPING: TOPOGRAPHIC COMPONENTCRITERIA FOR SUCCESSFUL APPLICATIONBASIC CRITERIAEXPANDED CRITERIAEFFECTIVE ON FORMALIN-FIXED, PARAFFIN-EFFECTIVE WITH DIFFERENT TYPES OFEMBEDDED TISSUES WITHOUT UNDUEFIXATIVESSPECIMENS SACRIFICEARCHIVAL TISSUE BLOCKS OF VARYINGSTORAGE AGEHISTOPATHOLOGIC &DETAILED SEPARATION OF MIXTURES OFTOPOGRAPHIC SAMPLING GUIDED BYCELLULAR COMPONENTSMICROSCOPIC CONSIDERATIONSSAMPLING OF NECROTIC, CRUSHED ORCONFINED TARGETING INVOLVING MINUTEOTHERWISE UNSUITABLE SITESBUT HIGHLY REPRESENTATIVE TISSUEBIOPSY SIZED SPECIMEN HANDLINGSAMPLINGCAPACITY FOR MULTIPLEX GENETIC ASSAYSWITHOUT THE NEED TO RECUT OR FURTHERSACRIFICE TISSUEMETHODOLOGICAL SIMPLICITYEXPEDITED ANALYSIS FOR TIMELY RESULTSHIGH EFFICACY WITH MINIMAL FAILURECLINICAL APPLICATIONRATECONSIDERATIONSCOMPATIBILITY WITH EXISTINGLABORATORY PRACTICESCOST EFFECTIVENESS OF TESTINGQUALITY ASSURANCE AND CONTROL